RESUMO
The absence of orthogonal aminoacyl-transfer RNA (tRNA) synthetases that accept non-L-α-amino acids is a primary bottleneck hindering the in vivo translation of sequence-defined hetero-oligomers and biomaterials. Here we report that pyrrolysyl-tRNA synthetase (PylRS) and certain PylRS variants accept α-hydroxy, α-thio and N-formyl-L-α-amino acids, as well as α-carboxy acid monomers that are precursors to polyketide natural products. These monomers are accommodated and accepted by the translation apparatus in vitro; those with reactive nucleophiles are incorporated into proteins in vivo. High-resolution structural analysis of the complex formed between one PylRS enzyme and a m-substituted 2-benzylmalonic acid derivative revealed an active site that discriminates prochiral carboxylates and accommodates the large size and distinct electrostatics of an α-carboxy substituent. This work emphasizes the potential of PylRS-derived enzymes for acylating tRNA with monomers whose α-substituent diverges substantially from the α-amine of proteinogenic amino acids. These enzymes or derivatives thereof could synergize with natural or evolved ribosomes and/or translation factors to generate diverse sequence-defined non-protein heteropolymers.
Assuntos
Aminoacil-tRNA Sintetases , Aminoacil-tRNA Sintetases/genética , Lisina/química , Aminoácidos , RNA de Transferência/genéticaRESUMO
Studies of the proteome would benefit greatly from methods to directly sequence and digitally quantify proteins and detect posttranslational modifications with single-molecule sensitivity. Here, we demonstrate single-molecule protein sequencing using a dynamic approach in which single peptides are probed in real time by a mixture of dye-labeled N-terminal amino acid recognizers and simultaneously cleaved by aminopeptidases. We annotate amino acids and identify the peptide sequence by measuring fluorescence intensity, lifetime, and binding kinetics on an integrated semiconductor chip. Our results demonstrate the kinetic principles that allow recognizers to identify multiple amino acids in an information-rich manner that enables discrimination of single amino acid substitutions and posttranslational modifications. With further development, we anticipate that this approach will offer a sensitive, scalable, and accessible platform for single-molecule proteomic studies and applications.
Assuntos
Proteoma , Proteômica , Aminoácidos/química , Aminopeptidases , Peptídeos/química , Proteômica/métodos , Semicondutores , Análise de Sequência de Proteína/métodosRESUMO
Although natural products and synthetic small molecules both serve important medicinal functions, their structures and chemical properties are relatively distinct. To expand the molecular diversity available for drug discovery, one strategy is to blend the effective attributes of synthetic and natural molecules. A key feature found in synthetic compounds that is rare in nature is the use of fluorine to tune drug behavior. We now report a method to site-selectively incorporate fluorine into complex structures to produce regioselectively fluorinated full-length polyketides. We engineered a fluorine-selective trans-acyltransferase to produce site-selectively fluorinated erythromycin precursors in vitro. We further demonstrated that these analogs could be produced in vivo in Escherichia coli on engineering of the fluorinated extender unit pool. By using engineered microbes, elaborate fluorinated compounds can be produced by fermentation, offering the potential for expanding the identification and development of bioactive fluorinated small molecules.
Assuntos
Produtos Biológicos , Policetídeos , Aciltransferases/metabolismo , Produtos Biológicos/química , Escherichia coli/genética , Escherichia coli/metabolismo , Flúor , Policetídeos/químicaRESUMO
We report that the recently introduced commercial strain of Vibrio natriegens (Vmax X2) supports robust unnatural amino acid mutagenesis, generating exceptional yields of soluble protein containing up to 5 noncanonical α-amino acids (ncAA). The isolated yields of ncAA-containing superfolder green fluorescent protein (sfGFP) expressed in Vmax X2 are up to 25-fold higher than those achieved using commercial expression strains (Top10 and BL21) and more than 10-fold higher than those achieved using two different genomically recodedEscherichia colistrains that lack endogenous UAG stop codons and release factor 1 and have been optimized for improved fitness and preferred growth temperature (C321.ΔA.opt and C321.ΔA.exp). In addition to higher yields of soluble protein, Vmax X2 cells also generate proteins with significantly lower levels of misincorporated natural α-amino acids at the UAG-programmed position, especially in cases where the ncAA is a moderate substrate for the chosen orthogonal aminoacyl tRNA synthetase (aaRS). This increase in fidelity implies that the use of Vmax X2 cells as the expression host can obviate the need for time-consuming directed evolution experiments to improve the selectivity of an aaRS toward highly desired but suboptimal ncAA substrates.
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Using cryo-electron microscopy (cryo-EM), we determined the structure of the Escherichia coli 70S ribosome with a global resolution of 2.0 Å. The maps reveal unambiguous positioning of protein and RNA residues, their detailed chemical interactions, and chemical modifications. Notable features include the first examples of isopeptide and thioamide backbone substitutions in ribosomal proteins, the former likely conserved in all domains of life. The maps also reveal extensive solvation of the small (30S) ribosomal subunit, and interactions with A-site and P-site tRNAs, mRNA, and the antibiotic paromomycin. The maps and models of the bacterial ribosome presented here now allow a deeper phylogenetic analysis of ribosomal components including structural conservation to the level of solvation. The high quality of the maps should enable future structural analyses of the chemical basis for translation and aid the development of robust tools for cryo-EM structure modeling and refinement.
Inside cells, proteins are produced by complex molecular machines called ribosomes. Techniques that allow scientists to visualize ribosomes at the atomic level, such as cryogenic electron microscopy (cryo-EM), help shed light on the structure of these molecular machines, revealing details of how they build proteins. Understanding how ribosomes work has many benefits, including the development of new antibiotics that can kill bacteria without affecting animal cells. Watson et al. used cryo-EM techniques with increased resolution to examine the ribosomes of the bacterium Escherichia coli in a higher level of detail than has been seen before. The results revealed two chemical modifications in proteins that form the ribosome that had not been observed in ribosomes previously. Additionally, a protein segment with a previously undescribed structure was identified close to the site where the ribosome reads the genetic instructions needed to make proteins. Further genetic analyses suggested these structures are in many related species, and may play important roles in how the ribosome works. Watson et al. were also able to see how paromomycin, an antibiotic used to treat parasitic infections, is positioned in the ribosome. The antibiotic interacts with a site near where the genetic code is read out, which might explain why certain changes to the antibiotic can interfere with its potency. Finally, the new ribosome structure reveals thousands of water molecules and metal ions that help keep the ribosome together as it produces proteins. This study shows the value of advances in cryo-EM technology and illustrates the importance of applying these techniques to other cell components. The results also reveal details of the ribosome useful for further research into this essential molecular machine.
Assuntos
Proteínas de Bactérias/ultraestrutura , Escherichia coli/ultraestrutura , RNA Bacteriano/ultraestrutura , Proteínas Ribossômicas/ultraestrutura , Ribossomos/ultraestrutura , Microscopia Crioeletrônica , RNA Mensageiro/ultraestrutura , RNA de Transferência/ultraestruturaRESUMO
By transplanting identity elements into E.â coli tRNAfMet , we have engineered an orthogonal initiator tRNA (itRNATy2 ) that is a substrate for Methanocaldococcus jannaschii TyrRS. We demonstrate that itRNATy2 can initiate translation inâ vivo with aromatic non-canonical amino acids (ncAAs) bearing diverse sidechains. Although the initial system suffered from low yields, deleting redundant copies of tRNAfMet from the genome afforded an E.â coli strain in which the efficiency of non-canonical initiation equals elongation. With this improved system we produced a protein containing two distinct ncAAs at the first and second positions, an initial step towards producing completely unnatural polypeptides inâ vivo. This work provides a valuable tool to synthetic biology and demonstrates remarkable versatility of the E.â coli translational machinery for initiation with ncAAs in vivo.
Assuntos
Aminoácidos/metabolismo , Biossíntese de Proteínas/fisiologia , Engenharia de Proteínas/métodos , HumanosRESUMO
Ribosome engineering has emerged as a promising field in synthetic biology, particularly concerning the production of new sequence-defined polymers. Mutant ribosomes have been developed that improve the incorporation of several nonstandard monomers including d-amino acids, dipeptides, and ß-amino acids into polypeptide chains. However, there remains little mechanistic understanding of how these ribosomes catalyze incorporation of these new substrates. Here, we probed the properties of a mutant ribosome-P7A7-evolved for better in vivo ß-amino acid incorporation through in vitro biochemistry and cryo-electron microscopy. Although P7A7 is a functional ribosome in vivo, it is inactive in vitro, and assembles poorly into 70S ribosome complexes. Structural characterization revealed large regions of disorder in the peptidyltransferase center and nearby features, suggesting a defect in assembly. Comparison of RNA helix and ribosomal protein occupancy with other assembly intermediates revealed that P7A7 is stalled at a late stage in ribosome assembly, explaining its weak activity. These results highlight the importance of ensuring efficient ribosome assembly during ribosome engineering toward new catalytic abilities.
Assuntos
Aminoácidos/genética , Escherichia coli/genética , Ribossomos/genética , Aminoácidos/química , Escherichia coli/química , Modelos Moleculares , Mutação , Peptídeos/química , Peptídeos/genética , Biossíntese de Proteínas , Ribossomos/químicaRESUMO
Here, we report that wild type Escherichia coli ribosomes accept and elongate precharged initiator tRNAs acylated with multiple benzoic acids, including aramid precursors, as well as malonyl (1,3-dicarbonyl) substrates to generate a diverse set of aramid-peptide and polyketide-peptide hybrid molecules. This work expands the scope of ribozyme- and ribosome-catalyzed chemical transformations, provides a starting point for in vivo translation engineering efforts, and offers an alternative strategy for the biosynthesis of polyketide-peptide natural products.
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The Center for Genetically Encoded Materials (C-GEM) is an NSF Phase I Center for Chemical Innovation that comprises six laboratories spread across three university campuses. Our success as a multi-institution research team demanded the development of a software infrastructure, GEM-NET, that allows all C-GEM members to work together seamlessly-as though everyone was in the same room. GEM-NET was designed to support both science and communication by integrating task management, scheduling, data sharing, and collaborative document and code editing with frictionless internal and public communication; it also maintains security over data and internal communications. In this Article, we document the design and implementation of GEM-NET: our objectives and motivating goals, how each component contributes to these goals, and the lessons learned throughout development. We also share open source code for several custom applications and document how GEM-NET can benefit users in multiple fields and teams that are both small and large. We anticipate that this knowledge will guide other multi-institution teams, regardless of discipline, to plan their software infrastructure and utilize it as swiftly and smoothly as possible.
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Polyketides are a large family of bioactive natural products synthesized by polyketide synthase (PKS) enzyme complexes predominantly from acetate and propionate. Given the structural diversity of compounds produced using these two simple building blocks, there has been longstanding interest in engineering the incorporation of alternative extender units. We have been investigating the mechanism of fluorinated monomer insertion by three of the six different modules of the PKS involved in erythromycin biosynthesis (6-deoxyerythronolide B synthase, DEBS) to begin understanding the contribution of different steps, such as enzyme acylation, transacylation, C-C bond formation, and chain transfer, to the overall selectivity and efficiency of this process. In these studies, we observe that inactivation of a cis-acyltransferase (AT) domain to circumvent its native extender unit preference leads concurrently to a change of mechanism in which chain extension with fluorine-substituted extender units switches largely to an acyl carrier protein (ACP)-independent mode. This result suggests that the covalent linkage between the growing polyketide chain and the enzyme is lost in these cases, which would limit efficient chain elongation after insertion of a fluorinated monomer. However, use of a standalone trans-acting AT to complement modules with catalytically deficient AT domains leads to enzyme acylation with the fluoromalonyl-CoA extender unit. Formation of the canonical ACP-linked intermediate with fluoromalonyl-CoA allows insertion of fluorinated extender units at 43% of the yield of the wild-type system while also amplifying product yield in single chain-extension experiments and enabling multiple chain extensions to form multiply fluorinated products.
Assuntos
Policetídeo Sintases/metabolismo , Policetídeos/metabolismo , Aciltransferases/metabolismo , Produtos Biológicos/metabolismo , Halogenação , Policetídeo Sintases/genética , Engenharia de ProteínasRESUMO
Over 30 years ago, GGDEF domain-containing enzymes were shown to be diguanylate cyclases that produce cyclic di-GMP (cdiG), a second messenger that modulates the key bacterial lifestyle transition from a motile to sessile biofilm-forming state. Since then, the ubiquity of genes encoding GGDEF proteins in bacterial genomes has established the dominance of cdiG signaling in bacteria. However, the observation that proteobacteria encode a large number of GGDEF proteins, nearing 1% of coding sequences in some cases, raises the question of why bacteria need so many GGDEF enzymes. In this study, we reveal that a subfamily of GGDEF enzymes synthesizes the asymmetric signaling molecule cyclic AMP-GMP (cAG or 3', 3'-cGAMP). This discovery is unexpected because GGDEF enzymes function as symmetric homodimers, with each monomer binding to one substrate NTP. Detailed analysis of the enzyme from Geobacter sulfurreducens showed it is a dinucleotide cyclase capable of switching the major cyclic dinucleotide (CDN) produced based on ATP-to-GTP ratios. We then establish through bioinformatics and activity assays that hybrid CDN-producing and promiscuous substrate-binding (Hypr) GGDEF enzymes are found in other deltaproteobacteria. Finally, we validated the predictive power of our analysis by showing that cAG is present in surface-grown Myxococcus xanthus. This study reveals that GGDEF enzymes make alternative cyclic dinucleotides to cdiG and expands the role of this widely distributed enzyme family to include regulation of cAG signaling.
Assuntos
Proteínas de Escherichia coli/metabolismo , Nucleotídeos Cíclicos/biossíntese , Fósforo-Oxigênio Liases/metabolismo , Deltaproteobacteria/enzimologia , Proteínas de Escherichia coli/química , Fósforo-Oxigênio Liases/química , Conformação ProteicaRESUMO
Cobamides, which include vitamin B12 (cobalamin), are a class of modified tetrapyrroles synthesized exclusively by prokaryotes that function as cofactors for diverse biological processes. Cobamides contain a centrally bound cobalt ion that coordinates to upper and lower axial ligands. The lower ligand is covalently linked to a phosphoribosyl moiety through an alpha-glycosidic bond formed by the CobT enzyme. CobT can catalyze the phosphoribosylation of a variety of substrates. We investigated the ability of CobT to act on either of two nitrogen atoms within a single, asymmetric benzimidazole substrate to form two isomeric riboside phosphate products. Reactions containing asymmetric benzimidazoles as substrates for homologues of CobT from different bacteria resulted in the production of distinct ratios of two isomeric products, with some CobT homologues favoring the production of a single isomer and others forming a mixture of products. These preferences were reflected in the production of cobamide isomers with lower ligands attached in different orientations, some of which are novel cobamides that have not been characterized previously. Two isomers of methoxybenzimidazolylcobamide were found to be unequal in their ability to support ethanolamine ammonia-lyase dependent growth in Salmonella enterica, suggesting that CobT's regiospecificity could be biologically important. We also observed differences in pKa, which can influence the reactivity of the cofactor and could contribute to these distinct biological activities. Relaxed regiospecificity was achieved by introducing a single point mutation in an active site residue of CobT. These new cobamide isomers could be used to probe the mechanisms of cobamide-dependent enzymes.
Assuntos
Proteínas de Bactérias/metabolismo , Biocatálise , Cobamidas/metabolismo , Modelos Moleculares , Complexos Multienzimáticos/metabolismo , Nucleotidiltransferases/metabolismo , Pentosiltransferases/metabolismo , Substituição de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Domínio Catalítico , Cobamidas/química , Cinética , Ligantes , Estrutura Molecular , Complexos Multienzimáticos/química , Complexos Multienzimáticos/genética , Mutagênese Sítio-Dirigida , Nucleotidiltransferases/química , Nucleotidiltransferases/genética , Pentosiltransferases/química , Pentosiltransferases/genética , Mutação Puntual , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Salmonella typhimurium/enzimologia , Salmonella typhimurium/crescimento & desenvolvimento , Salmonella typhimurium/metabolismo , Sinorhizobium meliloti/enzimologia , Especificidade da Espécie , Estereoisomerismo , Especificidade por Substrato , Veillonella/enzimologiaRESUMO
ortho-Quinone methides (ortho-QM) and para-quinone methides are generated by xenobiotic metabolism of numerous compounds including environmental toxins and therapeutic agents. These intermediates are highly electrophilic and have the potential to alkylate DNA. Assessing their genotoxicity can be difficult when all or some of their resulting adducts form reversibly. Stable adducts are most easily detected but are not necessarily the most prevalent products formed initially as DNA repair commences. Selective oxidation of ortho-QM-DNA adducts by bis[(trifluoroacetoxy)iodo]benzene (BTI) rapidly quenches their reversibility to prevent QM regeneration and allows for observation of the kinetic products. The resulting derivatives persist through standard enzymatic digestion, chromatography, and mass spectral analysis. The structural standards required for this approach have been synthesized and confirmed by two-dimensional NMR spectroscopy. The adducts of dA N(6), dG N1, dG N(2), and guanine N7 are converted to the expected para-quinol derivatives within 5 min after addition of BTI under aqueous conditions (pH 7). Concurrently, the adduct of dA N1 forms a spiro derivative comparable to that characterized previously after oxidation of the corresponding dC N3 adduct. By application of this oxidative quenching strategy, the dC N3 and dA N1 adducts have been identified as the dominant products formed by both single- and double-stranded DNA under initial conditions. As expected, however, these labile adducts dissipate within 24 h if not quenched with BTI. Still, the products favored by kinetics are responsible for inducing the first response to ortho-QM exposure in cells, and hence, they are also key to establishing the relationship between biological activity and molecular structure.
Assuntos
Adutos de DNA/química , Hidrocarbonetos Halogenados/química , Indolquinonas/química , Oligonucleotídeos/química , Alquilação , DNA/química , OxirreduçãoRESUMO
Physostigmine is a parasympathomimetic drug used to treat a variety of neurological disorders, including Alzheimer's disease and glaucoma. Because of its potent biological activity and unique pyrroloindole skeleton, physostigmine has been the target of many organic syntheses. However, the biosynthesis of physostigmine has been relatively understudied. In this study, we identified a biosynthetic gene cluster for physostigmine by genome mining. The 8.5â kb gene cluster encodes eight proteins (PsmA-H), seven of which are required for the synthesis of physostigmine from 5-hydroxytryptophan, as shown by in vitro total reconstitution. Further genetic and enzymatic studies enabled us to delineate the biosynthetic pathway for physostigmine. The pathway features an unusual reaction cascade consisting of highly coordinated methylation and acetylation/deacetylation reactions.